In recent years, as approaches for treating organs or tissues having functional failure or functional defects, the development of hybrid-type artificial organs (also referred to as biological artificial organs), and regenerative medical technologies, in which cultured cells and biocompatible materials are combined, have received much attention.
Currently, for instance, not less than 600,000 people are allegedly suffering from liver diseases in our country. In addition, about 50,000 patients have died a year because of liver diseases. Of those, about 1,000 patient deaths are due to acute liver failure and the remainder thereof is due to chronic hepatic insufficiency including hepatoma. A basic therapy for liver diseases such as hepatic insufficiency is liver transplantation. However, there is a large problem in that there is an insufficient number of offerers who are willing to donate their organs (i.e., donors). Therefore, the development of artificial livers has been demanded.
However, it is difficult to replace a total of 500 or more complicated liver functions with only an artificial means. As to an artificial liver, recently, a biological artificial liver using hepatic cells themselves has received much attention.
For the biological artificial liver, which is a representative example of a hybrid artificial organ, an extracorporeal circulation type therapeutic system is in the mainstream. The biological artificial liver carries out a therapeutic treatment by allowing a substance exchange through a plasma separator between a circuit on the body side for drawing out blood from all of the hepatic failure patients and circulating the blood and a circuit on the artificial liver module's side for carrying out a plasma circulation to metabolize and detoxify the plasma on the artificial liver module's side.
For such an artificial organ module, using dispersed cells is insufficient. That is, monolayer cultures which have been conventionally used for incubating cells cannot avoid loss or decrease of cellular functions. Thus, it is important to establish and use a multicellular aggregate body that resembles a living tissue.
From such a point of view, recently, a method of culturing an organ-like aggregate such as a spherical cell aggregate (spheroid) or a cylindrical cell aggregate (cylindroid) has been newly established, so that the high functional expression and long-term functional maintenance of cells will now be possible.
For example, as a method of culturing a spherical aggregate (spheroid), the inventors of the present invention have developed a method of forming a spheroid in a polymer base material such as a polyurethane foam (PUF) (JP10-29951A; and H. Ijima et al., “Tissue Engineering”, Vol. 4, No. 2, p. 213-226 (1998)). The PUF is made of a porous material having a main framework and a thin membrane beam structure. In addition, a certain degree of passage is formed between the pores of PUF, so a high density culture can be achieved under a good environment for substance exchange. When hepatic cells are cultured in the pores of PUF, about 200 hepatic cells gradually aggregate together to form many spherical multicellular aggregates (spheroids) each having a diameter of about 100 μm spontaneously. The inventors have succeeded in developing a short-term application type (about 10 days) biological artificial liver on a human clinical scale by means of a spherical aggregate (spheroid) using this culture method.
Furthermore, the inventors of the present invention have found that hepatic cells can be introduced into hollow fibers in a very dense state by means of a centrifugal force as a result of seeking out a compact artificial liver of a long-term application type. They have finally obtained an artificial module having a cell density of 2.4×107 cells/cm3 per module (see “Abstracts of 31st Summer Seminar Lectures in 2000 of Society of Fiber Science and Technology, Japan”, p. 115-118).
Furthermore, when an improvement in operability of the artificial liver at bedside and resolving the chronic lack of donors are taken into consideration, there is a need of a more compact artificial liver capable of maintaining its functions for a long time. Therefore, the inventors of the present invention have developed a higher density of hepatic cell aggregate (hepatic cell organoid) (JP 2002-247978 A).
However, those conventional cell aggregates (organoids) have been limited to spherical one(spheroid) or cylindrical ones (cylindroids) using perfect circle-shaped hollow fiber membranes. Of those, the cell aggregate of a hollow fiber membrane type is excellent in its handling properties or functionality as a device. On the other hand, if the inner diameter of the hollow fiber membrane used is too large, sufficient amounts of oxygen and nutrients would not diffuse to the cells located at the center of the cell aggregate, causing the necrosis thereof. As a result, there were problems in that one would not be able to use the cells being filled in the hollow fiber membrane efficiently without waste. Utilization efficiency of the cells becomes an extremely large problem when a cell source for making the cell aggregate (organoid) is rarely available like one of a brain-death donor origin. In contrast, if the inner diameter of the hollow fiber membrane is too small, the manufacture of uniform hollow fiber membranes and the modularization thereof becomes difficult. Furthermore, an airlock or the like can be caused and sometimes affected the operation of uniformly filling the cells.
On the other hand, when attention is paid to the structural aspects as a substance production device, a cell culturing device of a hollow fiber membrane type is known and can be roughly classified into a type of culture cells on the internal side of a hollow fiber membrane and supplying a culture medium to the external side thereof and the opposite type. In those cases, in general, a perfect circle-shaped hollow fiber membrane has been used, but sometimes other hollow fiber membranes having a different shape than a perfect circle has been used. For instance, JP 62-171678 A discloses that cells are incubated inside or outside a modified hollow fiber membrane having a fine extending in a longitudinal direction from the outer peripheral portion thereof. In addition, JP 63-233777 A discloses that cells are incubated outside the hollow fiber membrane having unevenness in a hollow portion.
The former case has a description that the hollow fiber membrane may be oval instead of a perfect circle. However, it was the hollow fiber membrane that requires a fin (finny protrusion) on the external portion of the membrane. A main purpose of the fin is to prevent close contact between the membranes to improve the dispersibility of a culture medium and cells. In the latter case, furthermore, the inner portion of the membrane was formed with irregularity in the longitudinal direction to prevent clogging of the membrane by causing turbulence in a culture medium flowing in the membrane.
As a complex, furthermore, U.S. Pat. No. 5,015,585 discloses a hollow fiber membrane having a so-called double structure in which another hollow fiber membrane is incorporated in a hollow fiber membrane. In this case, a gap between two hollow fiber membranes is made uniform for the purpose of keeping a survival rate of the cells filled in the gap. However, in obtaining the double-structured hollow fiber membrane, it is very difficult to make the structure uniform unless the raw materials of the membrane, physical properties of the membrane, size of the module, and the like are limited. Besides, in this case, the cells cannot be expected to be filled uniformly. In terms of this fact, to a large extent, it was not practical.
In this way, various studies have been made on the shapes of cell aggregates and the hollow fiber membrane type cell culturing devices. However, no technology has attempted to increase the efficiency of using the individual cells by fully working on the cross-sectional shape and diameter of the membrane on the basis of the cell aggregate formed in the hollow fiber membrane.